1. Field of Invention
The present invention relates to apparatus for carrying out patch clamp technique utilized to study ion transfer channels in biological membranes, and more particularly refers to such patch clamp apparatus having high through-put and utilizing only small test samples of compounds, only small amounts of liquid carrier, and capable of carrying out many tests in a short period of time with an individual membrane patch. The invention more broadly refers to a novel electrophysiology drug handling and application set up for screening of chemical substances or compounds, which provides high throughput, and which requires only low volume of solutions and samples to be tested. The invention also comprises several methods for utilizing the apparatus of the invention.
2. Prior Art
The general idea of electrically isolating a patch of membrane using a micropipette and studying the ion channels in that patch under voltage-clamp conditions was outlined by Neher, Sakmann, and Steinback in "The Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes", Pflueger Arch. 375; 219-278, 1978. They found that, by pressing a pipette containing acetylcholine (ACH) against the surface of a muscle cell membrane, they could see discrete jumps in electrical current that were attributable to the opening and closing of ACH-activated ion channels. However, they were limited in their work by the fact that the resistance of the seal between the glass of the pipette and the membrane (10-50 M.OMEGA.) was very small relative to the resistance of the channel (.about.10 G.OMEGA.). The electrical noise resulting from such a seal is inversely related to the resistance and was large enough to obscure the currents through ion channels, the conductances of which are smaller than that of the ACH channel. It also prohibited the clamping of the voltage in the pipette to values different from that of the bath due to the large currents through the seal that would result.
It was then discovered that by fire polishing the glass pipettes and applying gentle suction to the interior of the pipette when it made contact with the surface of the cell, seals of very high resistance (1-100 G.OMEGA.) could be obtained, which reduced the noise by an order of magnitude to levels at which most channels of biological interest can be studied and greatly extended the voltage range over which these studies could be made. This improved seal has been termed a "giga-seal", and the pipette has been termed a "patch pipette". For their work in developing the patch clamp technique, Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology and Medicine.
Ion channels are transmembrane proteins, which catalyze transport of inorganic ions across cell membranes. The ion channels participate in processes as diverse as generating and timing of action potentials, synaptic transmission, secretion of hormones, contraction of muscles, etc. Many drugs exert their specific effects via modulation of ion channels. Examples are antiepileptic compounds like phenytoin and lamotrigine, which block voltage dependent Na.sup.+ -channels in the brain, antihypertensive drugs like nifedipine and diltiazem, which block voltage dependent Ca.sup.2+ -channels in smooth muscle cells, and stimulators of insulin release like glibenclamide and tolbutamide, which block an ATP-regulated K.sup.+ -channel in the pancreas. In addition to chemically induced modulation of ion-channel activity, the patch clamp technique has enabled scientists to perform voltage dependent channel manipulations. These techniques include adjusting the polarity of the electrode in the patch pipette and altering the saline composition to moderate the free ion levels in the bath solution.
The patch clamp technique represents a major development in biology and medicine, since this technique allows measurement of ion flow through single ion channel proteins, and also allows the study of the single ion channel responses to drugs. In brief, in standard patch clamp technique, a thin (&lt;1 .mu.m in diameter) glass pipette is used. The tip of this patch pipette is pressed against the surface of the cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel proteins in a tiny patch of membrane. The activity of these channels can be measured electrically (single channel recording) or, alternatively, the patch clamp can be ruptured allowing measurements of the channel activity of the entire cell membrane (whole cell recording).
During both single channel recording and whole-cell recording, the activity of individual channel subtypes can be further resolved by imposing a "voltage clamp" across the membrane. Through the use of a feedback loop, the "voltage clamp" imposes a voltage gradient across the membrane, limiting overall channel activity and allowing resolution of discrete channel subtypes.
The time resolution and voltage control in such experiments are impressive, often in the msec or even .mu.sec-range. However, a major obstacle of the patch clamp technique as a general method in pharmacological screening has been the limited number of compounds that could be tested per day (typically no more than 1 or 2). Also, the very slow rate of solution change that can be accomplished around cells and patches may constitute a major obstacle.
A major limitation determining the throughput of the patch clamp technique is the nature of the feeding system, which leads the dissolved compound to perfused cells and patches. In usual patch clamp setups, cells are placed in large experimental chambers (0.2-2 ml), which are continuously perfused with a physiological salt solution. Compounds are then applied by changing the inlet to a valve connected to a small number of feeding bottles. However, a number of problems exist in the technique of the prior art. First, the number of different compounds is limited by the number of bottles that may be connected to the application system at one time. This number is usually less than 6. Second, the required volumes of the supporting liquid and the sample to be tested are critically high. Third, the time needed to change the solute composition around cells and patches is long for usual patch clamp experiments. Fourth, the introduction and application of compounds to be tested usually involves a significant degree of manual manipulation and interruption, thus jeopardizing the integrity of the cell/pipette connection.
The development of sophisticated systems for local application of compounds to activate neurotransmitter regulated channels, like the U-tube and other systems, reduces the effective application times to 10-100 msec, which is often acceptable. However, the volume of bath solution needed to be exchanged by these fast application systems is quite large and results in a limited capacity for screening multiple compounds per day. This presently limits the use of these procedures in medical industry. A major reason is the inflexibility and low capacity of the feeding systems which fill the U-tube. These are virtually identical to the systems used in conventional patch clamp experiments and therefore are still burdened with the inconveniences of these systems.